Fluorescent in vivo tracking of hematopoietic cells. Part I. Technical considerations

We report a new technology for in vivo tracking of hematopoietic cells, using fluorescent lipophilic probes. Because the probe is irreversibly bound in the lipids of the cell membrane; substantial numbers of dye molecules can be incorporated per cell and thus substantial signal to noise can be achieved. Although this technology can be used for all hematopoietic cells, these first findings are reported on red blood cells (RBCs) owing to the importance of the membrane to RBC function and integrity. We demonstrated that labeling 10% of the RBCs of a rabbit and reinjecting them into the animal makes possible the tracking of these cells at various times after injection. Furthermore, the labeling appears not to affect in vivo cell lifetime or cellular volume changes in response to hypotonic shock. The single cell fluorescence intensity of the labeled RBCs remains relatively constant for 60 days, and an immune response appears not to be generated against labeled cells. That labeled RBCs have lifetime kinetics in vivo, as shown in other studies, indicates that the membranes are functioning normally and are unaltered by the labeling technology. The technology we present is also applicable to white blood cells, bone marrow, and platelets.     

Regression of experimental Burkitt's lymphoma induced by Epstein-Barr virus-immortalized human B cells

Epstein-Barr virus (EBV)-immortalized human B cells survive only transiently when injected subcutaneously into athymic mice, whereas Burkitt's lymphoma cells give rise to progressively growing subcutaneous tumors. In this study, we tested whether these Burkitt's tumors could be induced to regress via a bystander effect induced by EBV-immortalized B cells. Simultaneous inoculation of EBV-immortalized B cells and Burkitt's lymphoma cells in the same subcutaneous site resulted in tumors that regressed with necrosis and scarring. Similarly, simultaneous inoculation of EBV-immortalized B cells and Burkitt's lymphoma cells in separate subcutaneous sites resulted in regression of a proportion of the Burkitt's tumors. Furthermore, most of the established human Burkitt's tumors regressed with necrosis and scarring after intratumor inoculations with EBV-immortalized B cells. The EBV-immortalized B cells continued to exert this antitumor effect even when killed with irradiation. The experimental approach to Burkitt's lymphoma treatment described here exploits the ability of athymic mice to reject EBV-immortalized B cells to target an effective antitumor response to malignant cells normally incapable of eliciting it.     

Rotational Atherectomy for Left Main Coronary Artery Disease in Octogenarians: Transradial Approach in a Tertiary Center and Literature Review

Objectives

The aim of this study was to appreciate the safety and effectiveness of transradial percutaneous coronary intervention (PCI) with rotational atherectomy for highly calcified left main coronary artery (LMCA) disease in octogenarians.

Background

Conventional surgery is still considered the preferred management for LMCA disease; but, when the lesion is severely calcified, and the patient is unsuitable for surgery, the interventional cardiologist faces a complex PCI traditionally approached by femoral access.

Methods

Between June 2004 and December 2010, octogenarians with calcified LMCA disease who were primary denied for surgical revascularization were enrolled. Procedural success and major adverse cerebral and cardiovascular events (MACCE) including death, nonfatal myocardial infarction, target lesion revascularization (TLR), or stroke during long‐term follow‐up were evaluated.

Results

Forty‐two consecutive patients≥80 years had undergone stenting for calcified LMCA disease (13 with rotational atherectomy, the “Rota” group, and 29 without rotational atherectomy, the “without Rota” group). Procedural success was good (92.3% vs. 96.6%, respectively, p = NS). Mean follow‐up time was 25.7 ± 21.4 and 28 ± 32.3 months, respectively. There was a TLR in 25% and 11.1%, respectively; p = NS. No difference was detected in terms of overall in‐hospital or long‐term mortality or MACCE.

Conclusion

Rotational atherectomy followed by stent implantation by transradial approach, when applied to heavily calcified lesions, appeared to be a safe and effective strategy for the treatment of LMCA disease in octogenarians who were refused for surgery. (J Interven Cardiol 2013;26:173–182)

     

Antioxidant,hepatoprotective and hypolipidemic effects of methanolic root extract of Cassia singueana in rats following acute and chronic carbon tetrachloride intoxication

ObjectiveTo evaluate in vivo antioxidant and hepatoprotective activities of the methanolic extract of the root of Cassia singueana in rats following acute and chronic carbon tetrachloride intoxication.MethodsMalondialdehyde (MDA), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and bilirubin as indices of liver damage and lipid peroxidation were detected in rats after intraperitoneal administration of extract (5 mg/kg).ResultsThe liver, kidney and heart showed significant reduction (P<0.05) in the levels of MDA from (0.18±0.04), (0.23±0.07) and (0.26±0.10) nmol/mg respectively in the CCl₄ control to (0.15±0.03), (0.17±0.04) and (0.17±0.07) nmol/mg protein in groups pre-treated with the extract for three days at 5 mg/kg). Similarly, compared to the CCl₄ control, significant reduction (P<0.05) in serum AST, ALT and bilirubin as well as in level of total cholesterol and MDA with concomitant increase in HDL cholesterol, superoxide dismutase and catalase levels when CCl₄-intoxicated rats were treated with Cassia singueana root extract for two weeks.ConclusionsThese results suggest that methanolic extract of Cassia singueana contain potent antioxidant compounds that can offer significant protection against hepatic and oxidative injuries.     

alpha-MSH acetylation in the pituitary gland of the sea bream (Sparus aurata L.) in response to different backgrounds, confinement and air exposure

MSH is a pituitary hormone derived by post-translational processing from POMC and involved in stress and background adaptation. N-terminal acetylation of MSH to monoacetyl alpha-MSH or diacetyl alpha-MSH increases the bioactivity of the peptide. The aim of this study was to characterize alpha-MSH acetylation in the sea bream (Sparus aurata L.) pituitary gland in response to the stressors air exposure and confinement, as well as in fish adapted for 15 days to a white, gray or black background. Pituitary homogenates were purified by reversed-phase HPLC (RP-HPLC). The alpha-MSH content of fractions was measured by RIA. Immunoreactive RP-HPLC fractions were further analyzed by electrospray mass spectrometry and the peptide sequence determined as SYSMEHFRWGKPV-NH2. In the pituitary gland of sea bream, des-, mono- and diacetyl alpha-MSH were identified. Then plasma alpha-MSH levels were measured in sea bream adapted to different backgrounds. Surprisingly, we found the highest plasma alpha-MSH levels in white-adapted as compared with black-adapted sea bream with intermediate values for gray-adapted fish. This observation is in contrast with results that have been obtained in eel, trout or terrestrial vertebrates. Next, des-, mono- and diacetyl alpha-MSH forms were measured in homogenates of the pituitary gland and in plasma of sea bream exposed to air, to confinement, or to different backgrounds. Monoacetyl alpha-MSH was the predominant form in all control and experimental groups. The lowest content of monoacetyl alpha-MSH relative to des- and diacetyl alpha-MSH was found in white-adapted fish. Levels of des- and diacetyl alpha-MSH forms were similar under all conditions. We observed that monoacetyl alpha-MSH is the most abundant isoform in the pituitary gland after background adaptation, confinement and air exposure, in sea bream. These data indicate that the physiologically most potent isoform of alpha-MSH may vary from species to species.     

氯沙坦减少高血压患者终点事件的研究(LIFE STUDY)

标　　题　应用ＡＴⅡ受体拮抗剂抗高血压治疗作　　者　ＫｊｅｌｄｓｅｎＳＥ,ＯｍｖｉｋＰ.　　参考文献　ＴｉｄｓｓｋｒＮｏｒＬａｅｇｅｆｏｒｅｎ,1996,116:504～507研究疾病　高血压病。目　　的　评估氯沙坦对心血管死亡率及非致死性心肌梗死和非脑卒中发生率的影响,同时评估该药对充血性心力衰竭或心绞痛所致的总死亡率和住院率的影响,以及长期应用氯沙坦对发生新诊断糖尿病的影响。设　　计　随机、三盲、对照研究。病人资料　8300例年龄55～80岁、收缩压在160～200ｍｍＨｇ、舒张压在95～115ｍｍＨｇ的高血压患者,ＥＣＧ上有左室…     

Transforming growth factor beta (TGF-beta), a potent inhibitor of erythropoiesis: neutralizing TGF-beta antibodies show erythropoietin as a potent stimulator of murine burst-forming unit erythroid colony formation in the absence of a burst-promoting activity

Transforming growth factor beta (TGF-beta) is a bifunctional regulator of the growth of myeloid progenitors and is here demonstrated to directly inhibit the growth of primitive erythroid progenitors by 95% to 100% regardless of the cytokines stimulating growth. Autocrine TGF- beta production of primitive hematopoietic progenitors has previously been reported. In the present study, a neutralizing TGF-beta antibody (anti-TGF-beta) added to serum-containing cultures, resulted in a 3-, 4- , and 25-fold increase in burst-forming unit erythroid (BFU-E) colony formation in response to interleukin-4 (IL-4) plus erythropoietin (Epo), SCF plus Epo, and IL-11 plus Epo, respectively. The growth of BFU-E progenitors has been suggested to require a burst-promoting activity in addition to Epo. Accordingly, we observed no BFU-E colony formation in serum-containing cultures in response to Epo alone. In contrast, 50 BFU-E colonies were formed when anti-TGF-beta was included in the culture. In serum-free cultures, Epo also stimulated BFU-E colony formation in the absence of other cytokines, whereas anti-TGF- beta had no effect on the number of colonies formed. Quantitation of TGF-beta 1 in serum by an enzyme-linked immunosorbent assay method showed predominantly the presence of precursor (latent) TGF-beta 1, but also showed active TGF-beta 1 at a concentration sufficient to potently inhibit erythroid colony formation. Thus, neutralization of active TGF- beta 1 in serum shows that Epo alone is sufficient to stimulate the growth of murine BFU-E progenitors.     

Immunophenotypic analysis of reticulocytes in paroxysmal nocturnal hemoglobinuria

The hematologic disorder paroxysmal nocturnal hemoglobinuria (PNH) occurs following an acquired somatic mutation in the Piga gene within a bone marrow stem cell. The progeny of this mutated cell cannot synthesize glycosylphosphatidylinositol (GPI) anchors, with a resultant deficiency in surface expression of all GPI-linked proteins. The protean clinical manifestations of PNH presumably result from the deficiency of these GPI-linked surface proteins. To explain the observation that neutrophils are affected at a significantly higher percentage than circulating erythrocytes and to analyze the proliferative rates of erythroid production in PNH, we studied 25 patients using flow cytometry. The fluorescent dye thiazole orange was used to detect reticulocytes, and CD59 monoclonal antibody was used to identify GPI-deficient cells. In contrast to the mature circulating erythrocytes, the percentage of abnormal reticulocytes was similar to the percentage of affected neutrophils. However, the vast majority of reticulocytes was completely GPI-deficient, ie, were type III cells, even in patients with only modest numbers of circulating type III erythrocytes. In addition, greater than 5% type II reticulocytes were identified in only 3 patients, although greater than 5% type II mature erythrocytes were identified in 10 of 25 patients. The results show that the erythroid and neutrophil bone marrow precursors have an equivalent proliferative advantage in PNH. The data also have important implications for the origin of type-II erythrocytes in PNH.